Diarrhoeagenic Escherichia Coli Pathotypes Research Articles

Pentavalent outer membrane vesicles immunized mice sera confers passive protection against five prevalent pathotypes of diarrhoeagenic Escherichia coli in neonatal mice

Diarrhoeagenic Escherichia coli (DEC) pathotypes are one of the major causative agents of diarrhoea induced childhood morbidity and mortality in developing countries. Licensed vaccines providing broad spectrum protection against DEC mediated infections are not available. Outer membrane vesicles (OMVs) are microvesicles released by gram-negative bacteria during the growth phase and contain multiple immunogenic proteins. Based on prevalence of infections, we have formulated a pentavalent outer-membrane vesicles (POMVs) based immunogen targeting five main pathotypes of DEC responsible for diarrhoeal diseases. Following isolation, OMVs from five DEC pathotypes were mixed in equal proportions to formulate POMVs and 10 µg of the immunogen was intraperitoneally administered to adult BALB/c mice. Three doses of POMVs induced significant humoral immune response against whole cell lysates (WCLs), outer membrane proteins (OMPs) and lipopolysaccharides (LPS) isolated from DEC pathotypes along with significant induction of cellular immune response in adult mice. Passive transfer of POMVs immunized adult mice sera protected neonatal mice significantly against DEC infections. Overall, this study finds POMVs to be immunogenic in conferring broad-spectrum passive protection to neonatal mice against five main DEC pathotypes. Altogether, these findings suggest that POMVs can be used as a potent vaccine candidate to ameliorate the DEC-mediated health burden.

Are non-lactose-fermenting Escherichia coli important diarrhoeal pathogens in children and adults?

Diarrhoeagenic Escherichia coli (DEC) remains one of the major causes of acute diarrhoea episodes in developing countries. The percentage of acute diarrhoea cases caused by DEC is 30-40 % in these countries. Approximately 10% of E. coli isolates obtained from stool specimens have been reported to be non-lactose-fermenting (NLF). The available literature is sparse regarding the pathogenicity of NLF E. coli causing infectious diarrhoea. We aimed to elucidate the importance of NLF E. coli in causing diarrhoea in both adults and children by detecting various DEC pathotypes among NLF E. coli in stool samples taken from gastroenteritis cases. A total of 376 NLF E. coli isolates from 3110 stool samples from diarrhoea/gastroenteritis patients were included in the study. Up to three NLF colonies that were not confirmed as Vibrio cholerae , Aeromonas spp., Salmonella spp. or Shigella spp., but were identified as E. coli using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), were carefully picked up from each MacConkey agar plate and then meticulously streaked onto freshly prepared, sterilized nutrient agar plates, and biochemical reactions were conducted. Multiplex PCR was conducted for the EAEC, EPEC, ETEC and EHEC pathotypes and PCR for the ipaH gene was conducted for EIEC. The disc diffusion method was used for antibiotic sensitivity testing. Using multiplex PCR and ipaH PCR, a total of 63 pathotypes of DEC were obtained, with EAEC being the most predominant (n=31) followed by EIEC (n=22), EPEC (n=8) and ETEC (n=2). To further differentiate EIEC from Shigella , additional biochemical tests were performed, including acetate utilization, mucate and salicin fermentation, and aesculin hydrolysis. Antimicrobial susceptibility testing (AST) showed that maximum resistance was seen against ciprofloxacin (82.5 %) followed by ampicillin (77.8 %) and cotrimoxazole (68.2 %), and minimum resistance was seen against ertapenem (4.8 %). In our study two pathotypes (EAEC, EIEC) were predominant among NLF E. coli and these were not only important aetiological agents in children, but also in adults. Our study also sheds light on the epidemiology of EIEC, which is one of the most neglected DEC pathotypes, as hardly any microbiological laboratories process NLF E. coli for EIEC.

Characterization of diarrhoeagenic Escherichia coli with special reference to antimicrobial resistance isolated from hospitalized diarrhoeal patients in Kolkata (2012-2019), India.

This study analyses the prevalence and antimicrobial resistance (AMR) of major diarrhoeagenic Escherichia coli (DEC) pathotypes detected in hospitalized diarrhoeal patients in Kolkata, India, during 2012-2019. A total of 8891 stool samples were collected from the Infectious Diseases Hospital, Kolkata and screened for the presence of enteric pathogens. Multiplex PCR identified the presence of DEC in 7.8% of the samples, in which ETEC was most common (47.7%) followed by EAEC (38.4%) and EPEC (13.9%). About 54% cases were due to sole DEC infections. Majority of the mixed DEC infections were caused by the Vibrio spp. (19.1%) followed by Rotavirus (14.1%) and Campylobacter spp. (8.4%). ETEC and EAEC were associated significantly with diarrhoea in children <5 years of age, whereas EPEC and also ETEC were prevalent in patients aged between 5 and 14 years. AMR profile showed high prevalence of multidrug resistance (MDR) among DEC (56.9%) in which 9% were resistant to antibiotics of six different antimicrobial classes. Screening of the AMR conferring genes of DEC showed the presence of blaCTX-M3 (30.2%) in highest number followed by blaTEM (27.5%), tetB (18%), sul2 (12.6%), strA (11.8%), aadA1 (9.8%), blaOXA-1 (9%), dfrA1 (1.6%) and blaSHV (1.2%). These findings highlighted the high prevalence of MDR in major DEC pathotypes that could be considered as the leading aetiological bacterial agent responsible for diarrhoea and suggests a significant public health threat. The results of this study can help to improve the understanding of the epidemiology of DEC infections in patients with diarrhoea. Monitoring of AMR surveillance needs special attention because the DEC isolates were highly resistant to commonly used antimicrobials in the treatment of diarrhoea.

Burden of Diarrhoeagenic Escherichia coli in Santa Rosa, Guatemala in active health-services surveillance during 2008-2009 and 2014-2015.

To describe the epidemiology of laboratory-confirmed Diarrhoeagenic Escherichia coli (DEC) cases from active facility-based surveillance in Guatemala. We collected clinical and risk factor data on enrolled patients (aged 0-52years) with acute diarrhoea at government healthcare facilities (1hospital and 6clinics) in Santa Rosa, Guatemala, during 2008-2009 and 2014-2015. Stool samples were analysed, E. coli identified through culture and biochemical tests, PCR amplification of genes encoding pathotype-specific virulence factors identified specific DEC pathotypes. Healthcare-seeking adjusted incidence rates were calculated. A total of 3041 diarrhoea cases were captured by surveillance (647hospitalisations (H), 2394clinic visits (CV)); general E. coli prevalence was 17.9%. DEC pathotypes were identified in 19% (n=95/497) and 21% (n=450/2113) in diarrhoea H and CV, respectively. Enteropathogenic E. coli (EPEC) was most frequently isolated (8.2% (n=41) in diarrhoea H, 12.0% (n=255) in diarrhoea CV), followed by ETEC (6.8% (n=34) in H, 6% (n=128) in CV) and STEC (0.6% (n=3) in H, 0.6% (n=13) in CV). We did not find evidence of a difference in severity between DEC and non-DEC diarrhoea. Incidence of DEC clinic visits and hospitalisations was 648.0 and 29.3, respectively, per 10,000 persons aged ≤5years and 36.8 and 0.4, respectively, per 10,000 persons aged >5years. DEC pathotypes, especially EPEC and ETEC, were detected frequently from patients presenting with diarrhoeal illness in Santa Rosa, Guatemala. Our findings suggest that preventive interventions should be prioritised for young children.

Diarrhoeagenic Escherichia coli isolated from children with acute diarrhoea at Rakai hospital, Southern Uganda.

Diarrhoeagenic Escherichia coli (DEC) is a leading cause of childhood diarrhoea. This study estimated the prevalence of DEC and DEC pathotypes among children with acute diarrhoea in Southern Uganda. A cross-sectional study was conducted on 267 children less than 5 years with acute diarrhoea, admitted to Rakai General Hospital in Southern Uganda. Faecal samples were collected from the children and processed for isolation of E. coli. The presence of DEC and the distribution of DEC pathotypes were determined by polymerase chain reaction. A total of 102 (38.2%, 102/267) children had DEC of various pathotypes - enteroaggregative E. coli (EAEC) (14.2%); enteropathogenic E. coli (EPEC) (6.7%); enterotoxigenic E. coli (ETEC) (6%); enteroinvasive E. coli (EIEC) (7.5%); enterohemorrhagic E. coli (EHEC) (3%); and cell-detaching E. coli (CDEC) (0.75%). The difference in the overall prevalence of DEC was not significant regarding HIV but individually, EAEC and CDEC were associated with HIV-positive status while ETEC was associated with HIV-negative status. DEC is prevalent in children with acute diarrhoea in Southern Uganda and its identification in children should be considered among strategies for combatting childhood diarrhoea in Africa.

Pathotyping and antimicrobial susceptibility testing of Escherichia coli isolates from neonatal calves.

Neonatal calf mortality is a major concern to livestock sector worldwide. Neonatal calf diarrhoea (NCD), an acute severe condition causes morbidity and mortality in calves. Amongst various pathogens involved in NCD, E. coli is considered as one of the major causes. The study was targeted to characterize E. coli isolates from neonatal calves for diarrhoeagenic Escherichia coli (DEC) types (pathotyping), antimicrobial resistance (AMR) profiling and to correlate with epidemiological parameters. From neonates, a total of 113 faecal samples were collected, out of that 308, lactose fermenting colonies were confirmed as E. coli. Pathotypable isolates (12.3%) were represented by STEC (6.1%), EPEC (2.9%), ETEC (1.9%), EAEC (0.9%) and EHEC (0.3%). Occurrence of STEC was more in non-diarrhoeic calves, whereas ETEC was observed more in diarrhoeic calves. EPEC occurrence was observed in both diarrhoeic and non-diarrhoeic calves. Fishers extract test showed no significant association for occurrence of DEC types to type of dairies, health status, species, breed, age and sex of neonatal calves. Two hundred and eighty isolates were tested for antimicrobial susceptibility. The isolates showed maximum resistance towards ampicillin (55.4%) followed by tetracycline (54.3%), while minimum resistance was observed towards meropenem (2.5%). Multidrug resistant E. coli isolates were found to be 139 (49.6%), and Extended-spectrum beta-lactamase (ESBL) producers were 120 (42.9%). DEC pathotypes like STEC, ETEC, EHEC and EAEC that are also multidrug resistant present in neonatal calves have zoonotic potential and hence are of public health significance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11259-021-09857-5.

Extended-spectrum beta-lactamase-producing strains among diarrhoeagenic Escherichia coli-prospective traveller study with literature review.

BackgroundAntibiotics are no longer the primary approach for treating all travellers’ diarrhoea (TD): most cases resolve without antibiotics and using them predisposes to colonization by multidrug-resistant bacteria. Data are accumulating on increasing resistance among TD pathogens, yet research into the most common agents, diarrhoeagenic Escherichia coli (DEC), remains limited.MethodsA total of 413 travellers to the (sub)tropics were analyzed for travel-acquired diarrhoeal pathogens and ESBL-PE. To identify ESBL-producing DEC, ESBL-producing E. coli (ESBL-EC) isolates were subjected to multiplex qPCR for various DEC pathotypes: enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC) and enterohaemorrhagic (EHEC) E. coli.For a literature review, we screened studies among travellers and locals in low- and middle-income countries (LMICs) on the frequency of ESBL-producing DEC, and among travellers, also DEC with resistance to ciprofloxacin, azithromycin, and rifamycin derivatives.ResultsOur rate of ESBL-EC among all DEC findings was 2.7% (13/475); among EAEC 5.7% (10/175), EPEC 1.1% (2/180), ETEC 1.3% (1/80) and EHEC (0/35) or EIEC 0% (0/5). The literature search yielded three studies reporting ESBL-EC frequency and thirteen exploring resistance to TD antibiotics among travel-acquired DEC. For EAEC and ETEC, the ESBL-EC rates were 10–13% and 14–15%, resistance to fluoroquinolones 0–42% and 0–40%, azithromycin 0–29% and 0–61%, and rifaximin 0% and 0–20%. The highest rates were from the most recent collections. Proportions of ESBL-producing DEC also appear to be increasing among locals in LMICs and even carbapenemase-producing DEC were reported.ConclusionESBL producers are no longer rare among DEC, and the overall resistance to various antibiotics is increasing. The data predict decreasing efficacy of antibiotic treatment, threatening its benefits, for disadvantages still prevail when efficacy is lost.

Antimicrobial resistance profiles of diarrhoeagenic Escherichia coli isolated from travellers returning to the UK, 2015-2017.

Introduction. Diarrhoeagenic Escherichia coli (DEC) are difficult to distinguish from non-pathogenic commensal E. coli using traditional culture methods. The implementation of PCR targeting specific virulence genes characteristic of the five DEC pathotypes, has improved the detection of DEC in faecal specimens from patients with symptoms of gastrointestinal disease.Aim. Antimicrobial resistance (AMR) profiles of 660 strains of DEC isolated between 2015 and 2017 from UK travellers reporting symptoms of gastrointestinal disease were reviewed to look for evidence of emerging AMR associated with travellers' diarrhoea.Methodology. All isolates of DEC were sequenced, and sequence type, serotype, pathotype markers and AMR profiles were derived from the genome data.Results. A travel history was provided for 54.1 % (357/660) of cases, of which 77.0 % (275/357) reported travel outside the UK within 7 days of onset of symptoms, and 23.0 % (82/357) reported no travel in that time frame. Of the 660 strains of DEC in this study, 265 (40.2 %) samples were identified as EAEC, 48 (7.3 %) as EIEC, 61 (9.2 %) were ETEC and 286 (43.3 %) were EPEC. EPEC caused the highest percentage of infections in children (40.6 %) whilst the highest proportion of cases reporting recent travel were infected with ETEC (86.1 %). There were 390/660 (59.0 %) isolates resistant to at least one antimicrobial on the panel tested (EIEC, 81.3 %; ETEC, n=65.6 %; EAEC, n=73.2 %; EPEC, 40.9 %) and 265/660 (40.2 %) were multidrug-resistant (EIEC, 33.3 %; ETEC, 32.8 %; EAEC, 56.2 %; EPEC, 28.0 %). Genes conferring resistance to the beta-lactams and fluroquinolones were highest in the EAEC pathotype, 56.6 and 60.7%, respectively.Conclusions. Increasing MDR, along with resistance to the fluroquinolones and the third-generation cephalosporins, in DEC causing travellers' diarrhoea provides further evidence for the need to restrict the use of antimicrobial agents and continuous monitoring.

Diarrhoeagenic Escherichia coli and Escherichia albertii in Brazil: pathotypes and serotypes over a 6-year period of surveillance.

Diarrhoeagenic Escherichia coli (DEC) is a leading cause of infectious diarrhoea worldwide. In recent years, Escherichia albertii has also been implicated as a cause of human enteric diseases. This study describes the occurrence of E. coli pathotypes and serotypes associated with enteric illness and haemolytic uremic syndrome (HUS) isolated in Brazil from 2011 to 2016. Pathotypes isolated included enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC). PCR of stool enrichments for DEC pathotypes was employed, and E. albertii was also sought. O:H serotyping was performed on all DEC isolates. A total of 683 DEC and 10 E. albertii strains were isolated from 5047 clinical samples. The frequencies of DEC pathotypes were 52.6% (359/683) for EPEC, 32.5% for EAEC, 6.3% for ETEC, 4.4% for EIEC and 4.2% for STEC. DEC strains occurred in patients from 3 months to 96 years old, but EPEC, EAEC and STEC were most prevalent among children. Both typical and atypical isolates of EPEC and EAEC were recovered and presented great serotype heterogeneity. HUS cases were only associated with STEC serotype O157:H7. Two E. albertii isolates belonged to serogroup O113 and one had the stx2f gene. The higher prevalence of atypical EPEC in relation to EAEC in community-acquired diarrhoea in Brazil suggests a shift in the trend of DEC pathotypes circulation as previously EAEC predominated. This is the first report of E. albertii isolation from active surveillance. These results highlight the need of continuing DEC and E. albertii surveillance, as a mean to detect changes in the pattern of pathotypes and serotypes circulation and provide useful information for intervention and control strategies.

A study of virulence and antimicrobial resistance pattern in diarrhoeagenic Escherichia coli isolated from diarrhoeal stool specimens from children and adults in a tertiary hospital, Puducherry, India

BackgroundEmergence of atypical enteropathogenic Escherichia coli (EPEC) and hybrid E. coli (harboring genes of more than one DEC pathotypes) strains have complicated the issue of growing antibiotic resistance in diarrhoeagenic Escherichia coli (DEC). This ongoing evolution occurs in nature predominantly via horizontal gene transfers involving the mobile genetic elements like integrons notably class 1 integron. This study was undertaken to determine the virulence pattern and antibiotic resistance among the circulating DEC strains in a tertiary care center in south of India.MethodsDiarrhoeal stool specimens were obtained from 120 children (< 5 years) and 100 adults (> 18 years), subjected to culture and isolation of diarrhoeal pathogens. Conventional PCR was performed to detect 10 virulence and 27 antimicrobial resistance (AMR) genes among the E. coli isolated.ResultsDEC infection was observed in 45 (37.5%) children and 18 (18%) adults, among which [18 (40%), 10 (10%)] atypical EPEC was most commonly detected followed by [6 (13.3%), 4 (4%)] ETEC, [5 (11.1%) 2 (2%)] EAEC, [(3 (6.6%), 0 (0%)] EIEC, [3 (6.6%), 0 (0%] typical EPEC, and [4 (8.8%), 1 (1%)] STEC, and no NTEC and CDEC was detected. DEC co-infection in 3 (6.6%) children, and 1(1%) adult and sole hybrid DEC infection in 3 (6.6%) children was detected. The distribution of sulphonamide resistance genes (sulI, sulII, and sulIII were 83.3 and 21%, 60.41 and 42.1%, and 12.5 and 26.3%, respectively) and class 1 integron (int1) genes (41.6 and 26.31%) was higher in DEC strains isolated from children and adults, respectively. Other AMR genes detected were qnrS, qnrB, aac(6’)Ib-cr, dhfr1, aadB, aac(3)-IV, tetA, tetB, tetD, catI, blaCTX, blaSHV, and blaTEM. None harbored qnrA, qnrC, qepA, tetE, tetC, tetY, ermA, mcr1, int2, and int3 genes.ConclusionsAtypical EPEC was a primary etiological agent of diarrhea in children and adults among the DEC pathotypes. Detection of high numbers of AMR genes and class 1 integron genes indicate the importance of mobile genetic elements in spreading of multidrug resistance genes among these strains.

Occurrence of diarrhoeagenic Escherichia coli virulence genes in water and bed sediments of a river used by communities in Gauteng, South Africa.

In most developing countries, especially in Southern Africa, little is known about the presence of diarrhoeagenic Escherichia coli (DEC) pathotypes in riverbed sediments. The present study sought to investigate the presence of DEC virulence genes in riverbed sediments of the Apies River, a river used by many communities in Gauteng, South Africa. Water and sediment samples were collected from the river between July and August 2013 (dry season) and also between January and February 2014 (wet season) following standard procedures. Isolation of E. coli was done using the Colilert®-18 Quanti-Tray® 2000 system. DNA was extracted from E. coli isolates using the InstaGene™ matrix from Bio-Rad and used as template DNA for real-time PCR. Water pH, temperature, dissolved oxygen, electrical conductivity and turbidity were measured in situ. Over 59% of 180 samples analysed were positive for at least one of the seven DEC virulence genes investigated. The eaeA gene was the most isolated gene (29.44%) while the ipaH gene the least isolated (8.33%). The ipaH gene (p = 0.012) and the ST gene (stIa, p = 0.0001, and stIb, p = 0.019) were positively correlated with temperature. The detection of diarrhoeagenic E. coli virulence genes in the sediments of the Apies River shows that the sediments of this river might not only be a reservoir of faecal indicator bacteria like E. coli but also pathogenic strains of this bacterium. These organisms could represent a public health risk for poor communities relying on this water source for various purposes such as drinking and recreational use. There is therefore an urgent need to monitor these DEC pathotypes especially in areas without adequate water supplies.

Genetic diversity and antibiogram profile of diarrhoeagenic Escherichia coli pathotypes isolated from human, animal, foods and associated environmental sources

IntroductionInfectious diarrhoea particularly due to pathogenic bacteria is a major health problem in developing countries, including India. Despite significant reports of diarrhoeagenic Escherichia coli (DEC) pathotypes around the globe, studies which address genetic relatedness, antibiogram profile and their correlation with respect to their isolation from different sources are sparse. The present study determines isolation and identification of DEC pathotypes from different sources, their genetic characterisation, antibiogram profile and their correlation if any.Materials and methodsA total of 336 samples comprising diarrhoeic stool samples from infants (n=103), young animal (n=106), foods (n=68) and associated environmental sources (n=59) were collected from Bareilly region of India. All the samples were screened by using standard microbiological methods for the detection of E. coli. The identified E. coli were then confirmed as DEC pathotypes using polymerase chain reaction–based assays. Those DEC pathotypes identified as Enteroaggregative E. coli (EAEC) were further confirmed using HEp-2 adherence assay. All the isolated DEC pathotypes were studied for their genetic diversity using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing was performed by using disc diffusion method as per Clinical Laboratory Standards Institute guidelines.Results and discussionOf the four DEC pathotypes investigated, EAEC was found to be the predominant pathogen with an isolation rate of 16.5% from infants, 17.9% from young animals, 16.2% from foods and 3.4% from the associated environmental sources. These EAEC isolates, on further characterisation, revealed predominance of ‘atypical’ EAEC, with an isolation rate of 10.7% from infants, 15.1% from young animals, 16.2% from foods, and 3.4% from the associated environmental sources. On PFGE analysis, discrimination was evident within DEC pathotypes as 52 unique pulsotypes were observed for 59 recovered DEC pathotypes. However, a few EAEC isolates were found to be clonal (clusters A, B, C, D, F, G, and H) irrespective of their source of isolation, suggests sharing and/or circulation among different sources. Further, a high antibiotic resistance pattern was observed among isolated DEC pathotypes as almost 86.4% of isolates were found to be resistant against ≥3 tested drugs.

Frequency and antimicrobial resistance of diarrhoeagenic Escherichia coli from young children in Iran

Diarrhoea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries. Diarrhoeagenic Escherichia coli (DEC) is an emerging agent among pathogens that cause diarrhoea. Between March 2011 and January 2012, a total of 600 stool specimens from children younger than 5 years of age (450 with and 150 without diarrhoea) were investigated for enteroaggregative E. coli (EAEC), enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) using PCR. Antimicrobial susceptibility testing was performed following Clinical and Laboratory Standards Institute guidelines. The prevalence of DEC pathotypes was 30.4% (137 patients) and 12% (18 patients) in the diarrhoea group and the control group, respectively. The most frequently isolated pathotype in diarrhoeal children was ETEC. This pathotype was detected significantly more often in children with diarrhoea (14.4%) than in children without diarrhoea (5.3%). EAEC and EPEC were detected with slightly higher frequencies in children with (8 and 4.2%, respectively) than in children without (4.6 and 2%, respectively) (P>0.05) diarrhoea. EHEC was only detected in children with diarrhoea (3.8%). Of the children from the diarrhoea group, 10% were colonized with more than one DEC pathotype. The DEC isolates exhibited high-level resistance to erythromycin (100%), azteronam (80.7%), amoxicillin (74.4%) and tetracycline (69.3%), and 86.4% of isolates were multidrug resistant. In conclusion, ETEC continues to be an important agent associated with diarrhoea in children from Tabriz, Iran.

Exhaustive isolation of diarrhoeagenic Escherichia coli by a colony hybridization method using hydrophobic grid-membrane filters in combination with multiplex real-time PCR

The present study aimed to develop a colony hybridization method for the exhaustive detection and isolation of diarrhoeagenic Escherichia coli (DEC) from samples containing numerous coliform bacteria. Digoxigenin-labelled DNA probes were designed to detect seven pathotypes of DEC based on type-specific genes. A total of 615 meat, food and faeces samples identified as DEC-positive by multiple real-time PCR for the virulence genes (eae, stx, elt, est, virB, aggR, afaB and astA) were analysed by a colony hybridization method, which involved filtering enrichment cultures through hydrophobic grid-membrane filters. DEC were isolated from 72.5% (446/615) of samples by the colony hybridization method but were only detected in 26.3% (162/615) of samples by a conventional culture method. The hybridization method was particularly effective for isolating low-level contaminants, such as enterotoxigenic and Shiga toxin-producing E. coli, which were isolated from 51.8% (58/112) of samples identified as positive by PCR for the enterotoxin genes, in contrast to only 4.5% (5/112) of samples analysed by the conventional method. The developed colony hybridization system allows for the efficient and simultaneous isolation of all DEC pathotypes. The colony hybridization system described here permits the sensitive isolation of DEC and represents a suitable tool for ecological investigations of DEC.